The applicant plans to study further the molecular basis of the structure of macrophage cortical cytoplasm. This study is relevent to the mechanism by which these cells move, undergo phagocytosis and secrete hydrolytic enzymes, processes which are fundamental in pulmonary defense and inflammation. The long term goal is to integrate the details of this cytoplasmic structure in the mechanism of these motile functions. There is evidence that the cytoplasm immediately beneath the membrane of motile cells is composed of a branching network of actin filaments. My previous work has established that a high molecular weight actin-binding protein (ABP) purified from macrophages crosslinks these actin fibers into such a network. My recnent characterization of the structure of ABP has shown that it is a flexible dimer with subunits linked "head-to-head" and that the sites which bind to actin are localized near the opposite ends, the "tails" of the dimers. These sites can bind actin monomers in solution and promote filament assembly, creating an actin filament network with perpendicular branches. The morphology resembles that of macrophage cortical cytoplasm and, the branch points appear to be secured by molecules of ABP. To prove these points and to learn more about ABP and its interaction with actin, the location of the actin binding sites on the ABP molecule will be defined by enzymatic digestion and the binding of actin nomomers to ABP molecules and binding fragments demonstrated. The actin binding domains of the ABP molecule will be characterized by biophysical techniques. Antibodies to this fragment will be prepared and morphological techniques will be used to definitively localize these sites on the parent molecule. Further, it will be shown directly by electron microscopy of detergent extracted macrophages and in reconstituted actin networks that ABP can interact with actin to form a 3-dimensional network and is indeed localized at the filament branch points both in vivo as well as in vitro.